Are red blood cells prokaryotic or eukaryotic?
eukaryotic
Is Trypanosoma cruzi prokaryotic or eukaryotic?
prokaryotic
What shape is Bacillus/bacilli?
rod shaped
What shape is coccus/cocci?
circular/round dot like
What shape is spirillum/Spirilla (pleural)
S" shaped
What does resolution mean?
the ability to distinguish between two adjacent objects
What color is Gram +?
Purplish/red
What color is Gram -?
Pink/fushia/colorless
When opening a culture tube, what is the purpose of flaming the mouth of the tube?
to kill any contaminants and to release a puff of air
Whats the purpose of flaming the inoculating loop?
to sterilize for aseptic technique
What are the reasons for heat-fixing the smear?
to kill the bacteria and it also helps the bacteria adhere to the slide
What are endospores?
resistive structures that resists adverse environbamental conditions
What is a vegetative cell?
bacteria cells that are metabolizing and reproducing rapidly
Why do ordinary stains like gram stain not work on acid fast bacteria?
these cells have a waxy lipid cell wall
What is the critical step in performing the acid fast stain?
successfully forcing the primary stain inside the cells by using heat
Name Two genera that contain species that are acid-fast.
mycobacterium & Nocardia
what general catagory of stains does capsule stain belong to?
negative stain
what are the two parts of a scientific name?
genus and species or specific epithet
What family of organic compounds does agar belong to?
carbohydrate
What is the purpose for agar in lab?
solidifying agent
What is the natural source of agar?
red seaweed or red algae
What is culturing bacteria?
actively growing
What is aseptic technique?
avoiding contaminants, preventing infection
What is a liquid medium called?
Broth
what is a solid medium called?
agar
What is growth medium?
supports growth of bacteria
What is a pure culture?
single species or kinds
what is a mixed culture?
two or more kinds
How big is a red blood cell? what can be seen by the naked eye?
7.5 microns/100 microns
Eukaryotic cell
larger and complex with organelles, nucleus
Prokaryotic cell
smaller, simple, no nucleus, no membrane bounded organelles
Inoculate
implantation of microorganism into or upon culture media
Incubate
placed under favorable conditions, temp control
Culturing
act of growing
Negative stain
stains the background
Simple stain
one dye and dyes the cells
Differential stain
uses more than one dye and stains different kinds of organisms different colors
Primary stain
Crystal Violet/Gram +/1 min
Secondary stain
iodine/Gram -/1min/ Mordant
What property of bacterial cell structure makes the Gram Stain possible?
different in cell wall thicknesses
What is Counterstain
safranin/1 min
Decolorizer
acetone/alcohol 5 seconds
What is the critical step of gram staining?
decolorizer is critical and the timing
Is acid-fast stain simple or differential stain?
differential
What is the purpose of acid fast?
high cell wall content and to identify Mycobacterium and Nocardia
What are two important diseases caused by acid fast bacteria?
TB and Leprosy
Mycobacterium tuberculosis and Mycobacterium leprae
how long can endospores last?
indefinitly
What are two genera of endospore forming bacteria that are medically important?
Bacillus and Clostridium
Non acid fast is?
blue
Acid Fast is?
Purple
Is mycobacterium acid fast or non acid fast? what color?
acid fast/ fuschia
What is a colony?
population of bacteria
What is a streak plate?
streaking for isolation
What effect does the prescence of capsules have one the pathogenicity of bacteria
sticks bacteria to surface, verolence factor, easily cause diseases makes it harder to phagocytose
What are the three basic cell shapes?
Coccus, Bacillus, and Coccibacillus.
Coccus
Cirlce in shape.
Bacillus
Rod shaped.
Coccibacillus
Circle/rod shaped.
Diplo
Two
Staphylo
Multiple Planes.
Strepto
Lines.
Spirochete
Flexible body.
Spirilum
Rigid body.
What is the purpose of heat fixing?
Kills the MO, and the MO adhere to the slide.
How do you sterilize the innoculating loop?
Sterilize by placing the innoculating loop inside of the bactcinerator for 7 - 10 seconds.
How do you know the innoculating loop is sterile?
The loop glows red for less than a second after being in the bactcinerator.
What is the purpose of a simple stain?
To determine size, shape and arrangements of bacteria.
What are the reagents for the simple stain?
Carolfuchsin, Methylene Blue, or Crystal Violet.
What is a differential stain?
The use of two or more dyes that react differently with different kinds/parts of bacteria.
What are examples of differential stains?
Acid-fast stain, Endospore stain, and Gram stain.
What is the procedure for a Negative Stain?
1. Create smear, do not heat fix.
2. Apply one to two drops of nigrosin, India ink, or eosin solution to the bacteria.
3. Slide another slide on top of the smear from one end to the other.
4. Air dry, do not heat fix.
When is Negative Staining used?
For bacteria that do not stain well like some spirochetes, or when it is desirable to confirm observations made on the shape and size of bacteria. It is also good for viewing capsules.
Why is heat fixing or harsh stains not used in the Negative Stain?
They can change the shape of the cells.
Why in the negative stain do the stains not penetrate the cell?
Because of the repulsion between the negative charge of the stains and the negative charge of the bacterial wall. Instead they produce a dark bacground so the bacteria appear as unstained cells with a clear area around them.
What are the Reagents of the negative stain?
Nigrosin, India Ink, or Eosin.
How do capsules influence virulence of a pathogenic bacteria?
The thicker the capsule on the bacteria, the higher the virulence the bacteria can hold. It varies with each bacterial species.
How does heat fixation effect the visibility of a capsule?
Heat fixing is not used in this procedure do to shrinkage, or destroying, is likely to occur and creates a clear zone around the bacterium, which can be mistaken for a capsule.
What is the Procedure for the Capsule Stain?
1. Smake smear.
2. Air dry ONLY.
3. Primary Stain: Crystal Violet. (stains cell purple, stains milk dark purple = dark background). 4 - 7 minutes.
4. Destain: Copper sulfate. (Do not remove dark purple background).
5. Blot with bibulous paper.
What are the Reagents for the Capsule Stain?
Crystal Violet Primary Stain, Destainer with Copper Sulfate which also acts as a counter stain.
What are the two uses for Copper Sulfate in the Capsule Stain?
A destainer as well as a counter stain.
What three items have been identified in Capsules?
Polysaccharides, polypeptides and glycoproteins.
What is the purpose of a Capsule for a bacteria?
Prevents the bacterium from dessecation (drying out), and protects it.
What are examples of bacterium that have capsules?
E. coli, K. pneumoniae, and b. megaterium.
What genera of bacteria are identified by the Acid-Fast Stain?
Mycobacteria and Norcardia.
What are two of the diseases caused by them bacteria that are identified in the Acid-Fast Stain?
Mycobacteria - TB/Leprosy, Norcardia - Rare Respiratory Infections, and Cryptosporidium.
What is the procedure for the Acid-Fast stain?
1. Make smear.
2. Add CarbolFuchsin - Primary Stain.
3. Heat fix to drive stain into the cell. (Carbolfucshin)
4. Decolorize with acid-alcohol drop by drop.
5. Rinse with water quickly.
6. Counterstain with Methylene blue for 2 minutes.
7. Rinse with wate
What will Acid Fast bacteria stain in colour?
Pink/Red.
What is Non Acid Fast bacteria stain in colour?
Blue/Purple.
What are the Reagents of the Acid-Fast stain?
Carbolfuchsin, Acid Alcohol, Methylene Blue.
What genera of bacteria produce spores?
Bacillus anthracis, Clostridium tetani, C. botulinium, C. difficile, and C. perfringens.
What are diseases caused by spore forming bacteria?
Bacillus anthracis - Anthrax, Clostridium tetani - Tetanus, C. botulinium - Botulism, C. difficile - C. dif, C. perfringens - Gangrene.
What is the function of spores?
A protective function in harsh living environments of some MO.
What causes the low permeability and high resistance of spores?
They have low permeability and high resistance due to multiple coats surrounding the spore.
What are the Reagents of Spore Stain?
Malchite Green - penetrates spore, Safranin - stains cell.
What is the procedure in the Spore Stain?
1. Make smear.
2. Primary stain - Malachite Green.
3. Heat over boiling water for ten minutes - forces M.G. into spore. (green spore at end of procedure).
4. Rinse with water.
5. Counterstain with Safranin. (60 - 90 seconds). (pink/red cells, spores stay
What is the procedure for the Gram Stain?
1. Make Smear.
2. Heat fix smears.
3. Primary Stain: Crystal Violet (C.V.) - Purple. (30 seconds).
4. Mordant: Grams Iodine (IKI) - Plugs P.G. layer in G+ cells, nothing in G-. (1 min)
5. Destain: Alcohol - acetone mix. MOST IMPORTANT. It too long G+ lose
What do Gram Positive cells appear in colour?
Blue.
What do Gram Negative cells appear in colour?
Pink/Red.
What cell structure takes up crystal violet to produce a Gram positive bacteria cell?
The cell wall is plugged by IKI (grams iodine), which keeps the C.V (crystal violet) in the cell wall in gram + bacteria, in gram - bacteria it has no effect.
What are the Reagents of a Gram Stain?
Crystal Violet, Grams Iodine (IKI), Alcohol Acetone mix, Safranin.
What is the most important step in the Gram Stain?
Decolorizing stage with Ethanol Acetone mix.
Why is the Decolorizing agent the most important step in the Gram Stain?
If done too long it can wash the Crystal Violet out of the Gram + cell wall, if too short it can leave the Crystal Violet in the Gram - cell.
Differential Media
Meda that distinguishes between different groups of bacteria and permit ID of MO based on their biological characteristics. i.e., colour change.
Selective Media
Favor the growth of a particular MO, while inhibiting the growth of another.
Mannitol Salt Agar (MSA)-What genus and species of bacteria is this media used to identify?
Staphylococcus, S. Aureus.
How does MSA media work and what are positive results?
This media can undergo fermentation, which lowers the pH of the media which causes a colour change from pink -> yellow (differential). It's high salt concentration inhibits the growth of many MO (selective).
Is MSA selective, differential, or both selective and differential?
Both selective and differential.
Eosin Methylene Blue Agar (EMB) and MacConkey Agar- What are the bacteria identified with these media?
Identifies G - bacteria, especially E. coli.
How does EMB media work and what are the results?
It inhibits the growth of G+ bacteria, allowing the G- bacteria to grow (selective). When E. coli grows, it produces a green metallic color (differential), which is due to the large amount of acids produced by fermentation of lactose.
Is EMB selective, differential, or both selective and differential?
Both selective and differential.
Anaerobic Bacteria
Do not like 02, 02 can be toxic depending on type of Anaerobe. Grow in bottom.
Aerobic Bacteria
Like 02, grow at top.
Facilitative Anaerobe
Grow throughout. Can be with and without 02.
What is the Special equipment required for growth of strict anaerobes?
They need to be placed in a anaerobic box, with the caps unscrewed 1 turn so that the air can be sucked out of it during the procedure. If the caps are left on then the air stays in the and test will be void.
Why is a Streak Plate used?
Used to get isolated, pure colonies by transfer in agar.
Where will the most and least bacteria be on the Streak Plate?
Most bacteria will be in zone 1, with most isolated colonies in zone 3 - 4.